Following murine injection, the ultrastructural loci of nanoparticles (NPs) containing lutetium-177 (Lu-177) conjugated to an anti-thrombomodulin antibody (mAb-201b) were determined. The results confirmed prior work localizing NPs using Single Photon Emission Computed Tomography (SPECT) scans. The in vivo pharmacokinetics of these NPs were also identified. mAb-201b antibodies are primarily attracted to the thrombomodulin, a membrane protein in the endothelium of the lung vasculature. SPECT images demonstrated NPs in the lungs, liver, spleen, and proximal small bowel. Prior injection of clodronate liposomes reduced the number of circulating macrophages, which, in turn, reduced NP phagocytosis. At 24 h after injection of NPs and after final SPECT imaging, the lungs, liver, spleen, and kidneys were harvested for transmission electron microscopy. Although some NPs were found in all four organs, 85% of the injected dose was localized in type I and type II pneumocytes. Small concentrations were found in secondary lysosomes in hepatocytes, in splenic macrophages, and in an intravascular macrophage in a kidney. Importantly, there was no apoptosis or necrosis in any of the tissues, highlighting the relative safety of the radionuclide NP, whose primary interaction with non-targeted organs/tissues is in the filtration process. In addition to validating the biodistribution results of the SPECT scans carried out in our prior work, this study is proof of principle that NPs conjugated with appropriate antibodies can target specific antigens in vivo. From a theranostic perspective, these results suggest that radioactive nanoconjugates labeled with proper antigens should be able to target and destroy a variety of cancers with minimal harm to the surrounding healthy cells.

Targeted radiotherapies maximize cytotoxicity to cancer cells. In this work, we describe the synthesis, characterization, and biodistribution of antibody conjugated gold-coated lanthanide phosphate nanoparticles containing 177Lu. [177Lu]Lu0.5Gd0.5(PO4)@Au@PEG800@Ab nanoparticles combine the radiation resistance of crystalline lanthanide phosphate for stability, the magnetic properties of gadolinium for facile separations, and a gold coating that can be readily functionalized for the attachment of targeting moieties. In contrast to current targeted radiotherapeutic pharmaceuticals, the nanoparticle-antibody conjugate can target and deliver multiple beta radiations to a single biologically relevant receptor. Up to 95% of the injected dose was delivered to the lungs using the monoclonal antibody mAb-201b to target the nanoparticles to thrombomodulin receptors. The 208 keV gamma ray from 177Lu decay (11%) can be used for SPECT imaging of the radiotherapeutic agent, while the moderate energy beta emitted in the decay can be highly effective in treating metastatic disease.

For targeted alpha therapy (TAT) with 225Ac, daughter radioisotopes from the parent emissions should be controlled. Here, we report on a second-generation layered nanoparticle (NP) with improved daughter retention that can mediate TAT of lung tumor colonies. NPs of La3+, Gd3+, and 225Ac3+ ions were coated with additional layers of GdPO4 and then coated with gold via citrate reduction of NaAuCl4. MAb 201b, targeting thrombomodulin in lung endothelium, was added to a polyethylene glycol (dPEG)-COOH linker. The NPs:mAb ratio was quantified by labeling the mAb with 125I. NPs showed 30% injected dose/organ antibody-mediated uptake in the lung, which increased to 47% in mice pretreated with clodronate liposomes to reduce phagocytosis. Retention of daughter 213Bi in lung tissue was more than 70% at one hour and about 90% at 24 hours postinjection. Treatment of mice with lung-targeted 225Ac NP reduced EMT-6 lung colonies relative to cold antibody competition for targeting or phosphate-buffered saline injected controls. We conclude that LnPO4 NPs represent a viable solution to deliver the 225Ac as an in vivo α generator. The NPs successfully retain a large percentage of the daughter products without compromising the tumoricidal properties of the α-radiation.

Colorectal cancer (CRC), is the second-leading cause of cancer-related deaths in the USA, affecting both men and women. Current projections show little or no change since the publication of a morbidity and mortality study in 2005. The projected number of new cases for 2008 is 154,000, and the projected number of CRC cancer deaths for 2008 is 53,000. The standard diagnostic paradigm is based on histopathology of either biopsy or surgical specimens. This article suggests a new paradigm for colon cancer diagnosis and staging using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS or IMS). IMS may identify potential tumors in normal tissue of cancer patients and predict those cancer patients who are at risk for recurrent cancer.

Purpose: MALDI imaging (IMS) demonstrated the same colon tumor proteins, gi| 119592539 and gi| 119592490, in consecutive patients. These proteins were present in the tumors and in normal satellite tissue. Similarly, putative colon cancer proteins were demonstrated in benign polyps. The proteome of normal tandem colon mucosa in patients with polyps or carcinomas is significantly different from that of patients without polyps or carcinomas. This raised several questions, is this evidence of metastatic disease or spread of tumor into normal satellite tissue and does the use of histopathology alone underestimate the extent of potential malignant disease? We hypothesized that histopathology in combination with IMS, may not only identify malignant disease in normal tissue, but also identify patients with field defects that are at risk for polyps and carcinomas. Methods: To test this hypothesis we examined tissue from 25 patients with normal colons at colonoscopy, and compared it with tissue from 25 patients with polyps and carcinomas respectively. Tissue included the tumor or polyp, and tandem proximal and distal normal control tissue. Contiguous histologic sections were obtained for histology (stained with H&E), IMS and protein extraction. MALDI images and protein masses were obtained on a Shimadzu Axima mass spectrometer. The third section was used for high pressure protein extraction and the extract was separated with nanoflow liquid chromatography, trypsinized and processed for protein identification with a nanoflow LCMS, Hitachi NanoFrontier. Results: IMS displayed the loci of proteins in the normal tissue of normal patients, normal tissue in patients with polyps or tumors, and in the polyps and tumors. The extraction experiments confirmed the proteins identified on IMS. There were clear differences in the normal mucosa proteome of the normal patients compared with the patients that had tumors or polyps. Putative carcinoma proteins were again identified in normal polyps. Conclusion: IMS and LCMS can identify protein biomarkers of mucosa at risk, putative carcinoma proteins in histologically normal polyps, and identify those tumors with concordant proteins in carcinomas and tandem normal mucosa.

Purpose: IBD is characterized by chronic intestinal inflammation in the absence of a recognized pathogen. Perinuclear antineutrophil cytoplasmic antibodies were identified in patients with ulcerative colitis (UC). IgG antibodies to cathepsin G were detectable in 38% of patients with Crohn's disease (CD), but negligible in patients with UC. Anti-Saccharomyces cerevisiae (ASCA) antibody was found in 50–70% of patients with CD, and 6–14% of patients with UC. ASCA is thought to be highly specific for CD. The bacterial sequence, I2, of the bacterium Pseudomonas fluorescens is found in 43% of involved CD colonic mucosa. IgA antibodies against I2 are present in 54% of patients with CD and 10% of patients with UC. C-reactive protein and thrombopoietin are elevated in IBD patients' serum. The surface molecule CD40 ligand is upregulated on IBD-derived platelets triggering chemokine release by CD40-bearing intestinal endothelial cells suggesting the participation of platelets in the inflammatory process. We hypothesized that matrix assisted laser desorption ionization mass spectrometry (MALDI), imaging MALDI (IMS) and liquid chromatograph mass spectrometry (LCMS) could demonstrate the loci of biomarker proteins in tissue microarrays, and define CD, UC, and non specific, NS, groups by their expressed proteins. Methods: The microarray includes the active inflammatory site, surrounding satellite, and normal control tissue from each patient. Contiguous histologic sections were obtained for IMS, 3, histology, 1, and protein extraction, 1. The paraffin was removed and the paraformaldehyde induced cross-links reversed by heating the sections to 90° C for 15 minutes. The histology sections were stained with hematoxylin and eosin. The MALDI matrices (sinapic acid, alpha cyano 4-hydroxy cinnamic acid, and 2, 5-dihydroxybenzoic acid) were sublimed respectively onto 3 IMS sections. IMS images were obtained on a Shimadzu Axima TOF2 mass spectrometer. The third section was used for high pressure protein extraction of individual microarray foci with a Pressure BioSciences Barocycler. The extract was separated with liquid chromatography and split into two aliquots: one for trypsin digest and bottom-up proteomics with a nanoflow LCMS, Hitachi NanoFrontier. The second intact protein aliquot was processed for top-down proteomics. Results: The high pressure extraction yielded new and larger numbers of proteins, decreased trypsin digest time from 12 hours to less than 45 minutes, and lower trypsin quantity for digest. Tissue microarray IMS displayed the loci of proteins, and separated the groups by their unique proteins. The LCMS experiments confirmed the proteins from IMS. Conclusion: IMS can separate IBD into three groups by their expressed proteins.

Micropipette tip (tip) pipetting accuracy and precision are functions of tip manufacture consistency, morphological variation, and retained inner tip wall particulate matter. Irregular tip inner wall surfaces and retained particulate matter cause pipetting inaccuracy. Washed and unwashed tips from seven manufacturers were compared using weight by difference (six), microscopic Luxol Fast Blue staining (seven), and matrix-assisted laser desorption/onization time-of-flight (MALDI-TOF) mass spectrometry (six). Photomicrographs revealed tip irregularity and inner wall retained particulate matter. Computer model analysis was used to identify tip irregularity and retained matter. These tests established the utility of a pipette solvent wash to increase the performance and the accuracy of tips and, thus, improve the MALDI mass spectra obtained.

Purpose: Imaging MALDI (IMS) demonstrated the same colon tumor proteins, gi|119592539 hCG1787564[Homo sapiens] Mass: 57590 and gi|119592490 hCG2040674[Homo sapiens] Mass: 108178, in consecutive patients. The proteins were present in the tumors and normal satellite tissue. The presence of these proteins in the tumor and normal tissue raised several questions. Is this evidence of metastatic disease or spread of tumor into normal satellite tissue? Are these proteins biomarkers of field cancerization or a field defect, e.g., age-related hypermethylation in normal colonic mucosa? Does the use of histopathology alone, underestimate the extent of potential malignant disease? We hypothesized that histopathology, in combination with IMS, may identify metaplastic disease beyond the recognized tumor. To test this hypothesis we examined tissue microarrays of multiple colon tumors. Methods: Tissue microarrays were constructed from colon carcinomas blocks. The tumor microarray from each patient included three components: the tumor, surrounding satellite tissue, and normal control tissue. Contiguous histologic sections were obtained for IMS,3, histology,1, and protein extraction,1. The paraffin was removed and the paraformaldeyde induced protein cross-links reversed by heating the sections to 90° C for 15 minutes. The histologic sections were stained with hematoxylin and eosin. The respective MALDI matrices: sinapic acid, alpha cyano 4-hydroxy cinnamic acid, and 2, 5-dihydroxybenzoic acids were applied by sublimation to the 3 IMS sections. MALDI images and protein masses were obtained on a Shimadzu Axima TOF2 mass spectrometer. The third section was used for high pressure protein extraction with a Pressure BioSciences Barocycler. The extract was separated with nanoflow Liquid Chromatograph Mass Spectrometry (LCMS) and split into two aliquots. One aliquot was trypsinized, and processed for bottom-up protein identification with a nanoflow LCMS, Hitachi NanoFrontier. The second intact protein aliquot was processed directly for top-down protein identification with LCMS. Results: The high pressure extraction provided novel results. The protein yield was increased. New and larger numbers of proteins were extracted. The trypsin digest time was decreased from 12 hours to 45 minutes, and less trypsin was required for the digest. Tissue microarray IMS displayed the loci of proteins in tumor, tumor satellite tissue, and normal tissue, and allowed separation of the tumors into groups distinguished by the unique proteins from each group. The extraction experiments confirmed the proteins identified on IMS. Conclusion: Tissue MALDI can separate colon tumors by protein profiles and identify those tumors with concordant proteins in tumor satellite tissue.

Proteins change rapidly in early brain ischemia. Their identification is likely key to the design of neuroprotective agents. In a recent model of focal cerebral infarction (Pevsner et al 2001; Eichenbaum et al 2002) we described MALDI-MS (matrix assisted laser desorption ionization mass spectrometry) identification of serum albumen and ß tubulin, a neuronal protein, minutes after the onset of ischemia in intact tissues (Pevsner et al 2006). In this study we were able to identify at least 6 proteins based on a BLAST query of the NCBI database using matrix (α-cyano hydroxyl cinnamic acid) alone on normal intact brain tissue. Mus musculus taxid 10090 gi|26337751|dbj|BAC32561.1| unnamed protein product [Mus m… gi|30520107|ref|NP_848815.1| G-rich RNA sequence binding f… gi|55976518|sp|Q8C5Q4|GRSF1_MOUSE G-rich sequence factor 1… gi|94406604|ref|XP_996413.1| PREDICTED: similar to lethal … gi|82998336|ref|XP_129160.6| PREDICTED: protein prenyltran… gi|94406148|ref|XP_993901.1| PREDICTED: hypothetical prote… These results demonstrate the utility of MALDI-MS for protein identification from cryostat sections without any trypsin digestion or extraction. This, with rapid brain removal and snap-freezing represents an ideal tool for dissecting dynamic processes in infarction, but also for identifying proteins in more slowly evolving processes such as neurodegenerative diseases.